CN102735740B - One-step rapid detection method of ochratoxin A by using electrochemical aptamer sensor - Google Patents
One-step rapid detection method of ochratoxin A by using electrochemical aptamer sensor Download PDFInfo
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Abstract
The invention relates to a one-step rapid detection method of ochratoxin A by using an electrochemical aptamer sensor, comprising the following steps: (1)polishing the surface of a bare gold electrode; (2)modifying the electrochemical aptamer sensor; (3) detecting an ochratoxin A standard concentration solution, and establishing a standard curve; and (4) carrying out quantitative determination on an actual sample solution containing ochratoxin A. According to the invention, the sensitivity of detecting ochratoxin is raised, the minimum detectable amount of ochratoxin A is 0.01 pg/mL, the cost of detecting the target toxin is greatly reduced, the operation is simple and convenient, and the one-step detection of ochratoxin A can be realized in 5-10 min.
Description
Technical field
The invention belongs to food inspection technical field, be specifically related to the detection method of ochratoxin A in food.
Background technology
Extensively there is occurring in nature in ochratoxin A, its strong toxicity is very big to the mankind and animals and plants infringement.Ochratoxin A is to be grown in the generations such as aspergillus on the crops such as grain (wheat, corn, barley, oat, rye, rice and broomcorn millet class etc.), peanut, vegetables (beans) and mould by multiple.Animal has been taken in after the feed going mouldy, and this toxin also may appear in the musculature of pig and hen etc.Ochratoxin A is mainly encroached on animal's liver and kidney.This toxin is mainly to cause kidney injury, and a large amount of toxin also may cause intestinal mucosa inflammation and the necrosis of animal.In animal experiment, observe its teratogenesis.
Ochratoxin A toxicity is very strong, and is extensively present in various food, cereal and secondary product thereof.In order to ensure the mankind's health, be necessary very much the detection research of ochratoxin A in conducting food.The content of ochratoxin A is conventionally very low, so to having relatively high expectations of detection means, the quilitative method that detects OTA has microtrabeculae method, and quantitative analysis method has thin-layered chromatography, high performance thin layer chromatography, HPLC, enzyme linked immunosorbent assay and fluorimetry etc.The advantage of thin layer chromatography is that method is simple, the reagent low price of use, but exist sensitivity poor, required reagent is various, and sense cycle is long, and reappearance is bad and cannot realize the shortcomings such as robotization, can not meet the requirement of modern measure.Chromatography can be qualitative, quantitative, but instrument is expensive, complicated operation and be not suitable for the detection of batch samples, and can not be for on-the-spot fast detecting.On-the-spot rapid screening method is more with enzyme linked immunosorbent assay.But this method is the compatible reaction based on Ag-Ab, using antibody as identification molecule.Because antibody is easily subject to the especially impact of temperature of external environment, limited the flexible Application of method.In addition, antibody preparation also needs through zoopery or cell experiment, and loaded down with trivial details time-consuming, preparation cost is high, and the cost of detection is also high.So be necessary to develop a kind of quick and convenient, testing cost is low, the novel detection method that detection sensitivity is high.
Summary of the invention
In order to solve the shortcoming that existing ochratoxin A testing cost is high, detection scheme is complicated, detection time is long, the invention provides a kind of method of utilizing galvanochemistry aptamer sensor one step fast detecting ochratoxin A.
Concrete technical solution is as follows:
In the situation that having ochratoxin A to exist, aptamers described in the present invention and ochratoxin A specific binding, the space structure that causes aptamer changes, further affect the electroactive substance methylene blue modified on aptamer and the speed of electrode surface electronics transmission, thereby affect the redox peak point current of electroactive substance methylene blue, the redox peak point current showing by electroactive substance methylene blue and the linear changing relation between ochratoxin A concentration, set up a step fast electrochemical sensor new detecting method of ochratoxin A.
The present invention utilizes the concrete operation step of galvanochemistry aptamer sensor one step fast detecting ochratoxin A method as follows:
(1). the polishing of naked gold electrode surfaces
Naked gold electrode sanding and polishing in the alumina powder aqueous solution of concentration 0.3 mmol/L is processed to 5min, in the alumina powder aqueous solution of concentration 0.05 mmol/L, sanding and polishing is processed 5 min again, then ultrasonic cleaning 5 min in ethanol solution, finally in deionized water for ultrasonic, clean 5 min, thoroughly remove non-specific adsorption at the alumina powder of naked gold electrode surfaces;
(2). the modification of aptamers electrochemical sensor:
Get three (β-chloroethyl) phosphate of 1mL concentration 1 mmol/L, join in the ochratoxin A aptamer solution that 99 mL concentration are 1mmol/L, under room temperature, 30 min are hatched in activation, obtain the ochratoxin A aptamer solution of activation; The ochratoxin A aptamer solution of getting 6 mL concentration 1mmol/L activation, drips the naked gold electrode surfaces after step (1) polishing, under 30 ℃ of conditions of temperature, hatches 2h, obtains the aptamer modified electrode of ochratoxin A; With the aptamer modified electrode of washed with de-ionized water ochratoxin A 2～3 times, the electrode that obtains cleaning; The electrode cleaning is immersed in the 6-sulfydryl hexanol of 200mL concentration 2mmol/L, and seal 4h under 30 ℃ of conditions of temperature, obtain the electrode that surface-closed is crossed, the electrode sealing by deionized water clean surface, obtains the galvanochemistry aptamer sensor that detects ochratoxin A for single stage method;
Ochratoxin A aptamer is ssDNA probe, the sequence of described ssDNA probe is GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA from 5 ' end to 3 ' end, the 3 ' end modified sulfydryl of described ssDNA probe, the 5 ' end modified electroactive substance methylene blue;
Concentration is that the ochratoxin A aptamer solution of 1mmol/L is the 3 ' end modified sulfydryl and the 5 ' end modified DNA aptamers probe solution of methylene blue;
(3). ochratoxin A normal concentration solution is detected to Criterion curve:
Using galvanochemistry aptamer sensor as working electrode, silver/silver chloride (Ag/AgCl) electrode is as contrast electrode, platinum electrode is as to electrode, be placed in the Tris-HCl damping fluid of 10 mL concentration 100 mmol/L, the pH value of Tris-HCl damping fluid is 7.4, and in Tris-HCl damping fluid, adding concentration is the ochratoxin A standard solution of 1mg/L, obtains the solution that contains ochratoxin A concentration 0.01pg/mL, insert described working electrode, incubated at room reaction 5～10min; Scanning square wave volt-ampere collection of illustrative plates, scanning noble potential is
0 V, electronegative potential is
-0.4 V, frequency 50 Hz, current potential increment is 0.001 V, amplitude is 0.05 V, obtains containing redox peak collection of illustrative plates and the redox peak current thereof that ochratoxin A concentration is 0.01pg/mL;
In like manner, containing ochratoxin A concentration 0.01
pg/mLsolution in then to add concentration be final concentration that 1mg/L ochratoxin A standard solution obtains respectively the containing ochratoxin A solution A that is 0.1pg/mL, the final concentration that contains ochratoxin A is 1 pg/mL solution B, the final concentration that contains ochratoxin A be the solution D that the solution C of 10 pg/mL, the final concentration that contains ochratoxin A are 100pg/mL, described solution A, solution B, solution C and solution D are four kinds of titers; Working electrode is inserted respectively in four kinds of titers successively, react respectively 5～10min; Scan respectively the square wave volt-ampere collection of illustrative plates of four kinds of titers and record corresponding redox peak point current; Using redox peak to peak current value corresponding to the solution that contains ochratoxin A concentration 0.01pg/mL and four kinds of titers as ordinate, using the concentration of ochratoxin A solution that contains 0.01pg/mL and the concentration of four kinds of standard ochratoxin A solution as horizontal ordinate, set up the typical curve that ochratoxin single stage method detects;
(4). the actual sample solution containing ochratoxin A is quantitatively detected:
Galvanochemistry aptamer sensor as working electrode is immersed in the Tris-HCl damping fluid that 10 mL concentration are 100 mmol/L, the pH value of Tris-HCl damping fluid is 7.4, in Tris-HCl damping fluid, add the ochratoxin A solution that contains unknown concentration, reaction 5～10 min, scanning square wave volt-ampere collection of illustrative plates, scanning noble potential is
0 V, electronegative potential is
-0.4 V, frequency 50 Hz, current potential increment is 0.001V, amplitude 0.05 V; Obtain the redox peak to peak current value that certain concentration ochratoxin is corresponding, by described ochratoxin single stage method examination criteria curve, calculate the concentration of ochratoxin A in the ochratoxin A solution of described unknown concentration.Detection ochratoxin A biology sensor used is responsive to target ochratoxin A and its concentration is converted to the instrument that electric signal detects.To make identification probe (comprising the bioactivators such as enzyme, antibody, antigen, microorganism, cell, tissue, nucleic acid) and suitable physics and chemistry transducer (as oxygen electrode, photosensitive tube, field effect transistor, piezoelectric crystal etc.) and analysis tool or the system of signal amplifying apparatus formation by immobilized biological sensitive materials.Aptamer is that the system planning technology screening by index concentration part comes.Compare with antibody, aptamer have advantages of easily synthetic, easily modify, easily fixing, can Reusability and long preservation.Galvanochemistry aptamer sensor plays an important role in quick test, and it combines the high sensitivity of electrochemical signals conduction and the high-affinity of aptamer.Galvanochemistry aptamer sensor prepare easy, easy modification, sensitive height, testing expense low, be suitable for the advantages such as onlineization, good stability and combining target thing scope are wide.
The invention solves ochratoxin A testing cost high, detection scheme is complicated, the shortcoming that detection time is long.The present invention adopts the aptamer of can specificity knowing ochratoxin A to be applied to electrochemical sensor, improved greatly the sensitivity detecting, reduced testing cost, and detect fast, only needing a step, do not need complicated detection means and expensive instrument, is a kind of quick and convenient, testing cost is low, the novel step in-situ check and test method that detection sensitivity is high.
Beneficial effect imbody of the present invention is in the following areas in sum:
1. adopt can be with the aptamer probe of ochratoxin A specific binding as identification probe in the present invention, build relevant sensing interface as biology sensor for detection of, improved the sensitivity that detects ochratoxin, the minimum ochratoxin A that can detect 0.01 pg/mL;
2. the present invention, as a kind of method of the Electrochemical Detection ochratoxin A based on aptamer, greatly reduces the cost that detects target toxin, simple to operation;
3. the present invention's one step fast detecting ochratoxin A, after prepared by galvanochemistry aptamer sensor, only needs 5～10 min just can realize a step and detects ochratoxin A.
Accompanying drawing explanation
Fig. 1 is that ochratoxin A standard solution detects the canonical plotting obtaining.
Embodiment
Below in conjunction with embodiment, the present invention is further described.
Embodiment:
(1) polishing of naked gold electrode surfaces
Naked gold electrode sanding and polishing in the alumina powder aqueous solution of concentration 0.3 mmol/L is processed to 5 min, in the alumina powder aqueous solution of concentration 0.05 mol/L, sanding and polishing is processed 5 min again, then ultrasonic cleaning 5 min in ethanol solution, finally in deionized water for ultrasonic, clean 5 min, thoroughly remove non-specific adsorption at the alumina powder of naked gold electrode surfaces;
(2) modification of aptamers electrochemical sensor:
Get three (β-chloroethyl) phosphate of 2 mL concentration 1 mmol/L, joining 198 mL concentration is in the ochratoxin A aptamer solution of 1 mmol/L, under room temperature, 30 min are hatched in activation, obtain the ochratoxin A aptamer solution of activation; The ochratoxin A aptamer solution of getting 12 mL concentration 1 mmol/L activation, drips the naked gold electrode surfaces after polishing in (1), under 30 ℃ of conditions of temperature, hatches 2 h, obtains the aptamer modified electrode of ochratoxin A; With the aptamer modified electrode of washed with de-ionized water 2～3 times, the electrode that obtains cleaning; The electrode cleaning is immersed in the 6-sulfydryl hexanol of 200 mL concentration 2 mmol/L, and seal 4 h under 30 ℃ of conditions of temperature, obtain the electrode that surface-closed is crossed, the electrode sealing by deionized water clean surface, obtains the galvanochemistry aptamer sensor that detects ochratoxin A for single stage method;
With the aptamer of ochratoxin A specific binding be ssDNA probe, the sequence of this single stranded DNA is GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA from 5 ' end to 3 ' end, the 3 ' end modified sulfydryl of this single stranded DNA, the 5 ' end modified electroactive substance methylene blue;
Described and aptamer solution ochratoxin A specific binding are the 3 ' end modified sulfydryl and the 5 ' end modified DNA aptamers probe solution of methylene blue;
(3) ochratoxin A normal concentration solution is detected to Criterion curve:
Using galvanochemistry aptamer sensor as working electrode, silver/silver chloride (Ag/AgCl) electrode is as contrast electrode, platinum electrode is as to electrode, be placed in the Tris-HCl damping fluid of 10 mL concentration 100 mmol/L, the pH value of Tris-HCl damping fluid is 7.4, in Tris-HCl damping fluid, adding concentration is the ochratoxin A standard solution of 1 mg/L, obtain containing the solution that ochratoxin A concentration is 0.01 pg/mL, after working electrode is inserted, incubated at room reaction 5～10min; Scanning square wave volt-ampere collection of illustrative plates, scanning noble potential is 0 V, electronegative potential is-0.4 V, frequency 50 Hz, current potential increment is 0.001 V, and amplitude is 0.05 V, obtains containing redox peak collection of illustrative plates and the redox peak current thereof that ochratoxin A concentration is 0.01pg/mL;
In like manner, in the solution that contains ochratoxin A concentration 0.01 pg/mL, then adding concentration is that final concentration that 1 mg/L ochratoxin A standard solution obtains respectively containing ochratoxin A is that the solution A of 0.1 pg/mL, the final concentration that contains ochratoxin A are that the solution B of 1 pg/mL, the final concentration that contains ochratoxin A are the solution D that the solution C of 10 pg/mL, the final concentration that contains ochratoxin A are 100pg/mL, and described solution A, solution B, solution C and solution D are four kinds of titers; Working electrode is inserted respectively in four kinds of titers successively, react respectively 5～10min; Scan respectively the square wave volt-ampere collection of illustrative plates of four kinds of titers and record corresponding redox peak point current; Redox peak to peak current value corresponding to the five kinds of ochratoxin A concentration of usining be as ordinate, usings the concentration of ochratoxin A as horizontal ordinate, sets up the typical curve that ochratoxin single stage method detects;
(4) the red wine sample solution containing ochratoxin A is quantitatively detected:
Galvanochemistry aptamer sensor as working electrode is immersed in the Tris-HCl damping fluid that 10 mL concentration are 100 mmol/L, the pH value of Tris-HCl damping fluid is 7.4, the ochratoxin A red wine solution that adds 1 mL to contain unknown concentration in Tris-HCl damping fluid, reaction 5～10 min, scanning square wave volt-ampere collection of illustrative plates, scanning noble potential is
0 V, electronegative potential is
-0.4 V, frequency 50 Hz, current potential increment is 0.001V, amplitude 0.05 V; Obtain containing the redox peak to peak current value that certain concentration ochratoxin A red wine sample is corresponding, by described ochratoxin A single stage method examination criteria curve, calculate the concentration of ochratoxin A in red wine sample.
Claims (1)
1. utilize the method for galvanochemistry aptamer sensor one step fast detecting ochratoxin A, it is characterized in that the concrete operation step of described method is as follows:
(1). the polishing of naked gold electrode surfaces
Naked gold electrode sanding and polishing in the alumina powder aqueous solution of concentration 0.3mmol/L is processed to 5min, in the alumina powder aqueous solution of concentration 0.05mmol/L, sanding and polishing is processed 5min again, then ultrasonic cleaning 5min in ethanol solution, finally in deionized water for ultrasonic, clean 5min, thoroughly remove non-specific adsorption at the alumina powder of naked gold electrode surfaces;
(2). the modification of aptamers electrochemical sensor:
Get three (β-chloroethyl) phosphate of 1mL concentration 1mmol/L, joining 99mL concentration is in the ochratoxin A aptamer solution of 1mmol/L, and under room temperature, 30min is hatched in activation, obtains the ochratoxin A aptamer solution of activation; Get the ochratoxin A aptamer solution of 6mL concentration 1mmol/L activation, drip the naked gold electrode surfaces after step (1) polishing, under 30 ℃ of conditions of temperature, hatch 2h, obtain the aptamer modified electrode of ochratoxin A; With the aptamer modified electrode of washed with de-ionized water ochratoxin A 2～3 times, the electrode that obtains cleaning; The electrode cleaning is immersed in the 6-sulfydryl hexanol of 200mL concentration 2mmol/L, and seal 4h under 30 ℃ of conditions of temperature, obtain the electrode that surface-closed is crossed, the electrode sealing by deionized water clean surface, obtains the galvanochemistry aptamer sensor that detects ochratoxin A for single stage method;
Ochratoxin A aptamer is ssDNA probe, the sequence of described ssDNA probe is GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA from 5 ' end to 3 ' end, the 3 ' end modified sulfydryl of described ssDNA probe, the 5 ' end modified electroactive substance methylene blue;
Concentration is that the ochratoxin A aptamer solution of 1mmol/L is the 3 ' end modified sulfydryl and the 5 ' end modified DNA aptamers probe solution of methylene blue;
(3). ochratoxin A normal concentration solution is detected to Criterion curve:
Using galvanochemistry aptamer sensor as working electrode, silver/silver chloride (Ag/AgCl) electrode is as contrast electrode, platinum electrode is as to electrode, be placed in the Tris-HCl damping fluid of 10mL concentration 100mmol/L, the pH value of Tris-HCl damping fluid is 7.4, and in Tris-HCl damping fluid, adding concentration is the ochratoxin A standard solution of 1mg/L, obtains the solution that contains ochratoxin A concentration 0.01pg/mL, insert described working electrode, incubated at room reaction 5～10min; Scanning square wave volt-ampere collection of illustrative plates, scanning noble potential is 0V, electronegative potential is-0.4V, frequency 50Hz, current potential increment is 0.001V, amplitude is 0.05V, obtains containing redox peak collection of illustrative plates and the redox peak current thereof that ochratoxin A concentration is 0.01pg/mL;
In like manner, then the solution D that the solution B that the final concentration that to add concentration in the solution that contains ochratoxin A concentration 0.01pg/mL be final concentration that 1mg/L ochratoxin A standard solution obtains respectively the containing ochratoxin A solution A that is 0.1pg/mL, contain ochratoxin A is 1pg/mL, solution C that the final concentration that contains ochratoxin A is 10pg/mL, the final concentration that contains ochratoxin A are 100pg/mL, described solution A, solution B, solution C and solution D are four kinds of titers; Working electrode is inserted respectively in four kinds of titers successively, react respectively 5～10min; Scan respectively the square wave volt-ampere collection of illustrative plates of four kinds of titers and record corresponding redox peak point current; Using redox peak point current corresponding to the solution that contains ochratoxin A concentration 0.01pg/mL and four kinds of titers as ordinate, using the concentration of ochratoxin A solution that contains 0.01pg/mL and the concentration of four kinds of standard ochratoxin A solution as horizontal ordinate, set up the typical curve that ochratoxin A one step detects;
(4). the actual sample solution containing ochratoxin A is quantitatively detected:
Galvanochemistry aptamer sensor as working electrode is immersed in the Tris-HCl damping fluid that 10mL concentration is 100mmol/L, the pH value of Tris-HCl damping fluid is 7.4, in Tris-HCl damping fluid, add the ochratoxin A solution that contains unknown concentration, reaction 5～10min, scanning square wave volt-ampere collection of illustrative plates, scanning noble potential is 0V, electronegative potential is-0.4V, frequency 50Hz, current potential increment is 0.001V, amplitude 0.05V; Obtain the redox peak point current that certain concentration ochratoxin A is corresponding, by described ochratoxin A one step examination criteria curve, calculate the concentration of ochratoxin A in the ochratoxin A solution of described unknown concentration.
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| CN104630869B (en) * | 2015-01-22 | 2017-07-14 | 江南大学 | A kind of DNA sensor for detecting staphylococcus aureus and its preparation and application |
| CN104965073A (en) * | 2015-05-25 | 2015-10-07 | 东华大学 | Electrochemical nucleic acid aptamer biosensor for detecting ibuprofen, and production method thereof |
| CN105567836A (en) * | 2016-02-01 | 2016-05-11 | 河南省农业科学院农业质量标准与检测技术研究所 | Signal amplification nucleic acid aptamer sensor for determining aflatoxin B1 and preparation method of sensor |
| CN106596677A (en) * | 2016-12-27 | 2017-04-26 | 江苏大学 | Preparation method of jettisonable aptamer sensor for OTA sensitivity detection |
| CN107389923A (en) * | 2017-08-01 | 2017-11-24 | 甘肃农业大学 | A kind of preparation method for exempting from mark type aptamer sensor and the method with the sensor quick detection ochratoxin A |
| CN108181371B (en) * | 2017-12-14 | 2020-07-17 | 江西农业大学 | Electrochemical sensing analysis method for simply and rapidly detecting ochratoxin A in food |
| CN110734962B (en) * | 2019-11-06 | 2021-10-19 | 江苏开放大学（江苏城市职业学院） | Method for detecting food toxin based on aptamer |
| CN111024791B (en) * | 2019-12-26 | 2021-01-01 | 中国科学院生态环境研究中心 | Electrochemical sensor and method for detecting ochratoxin A |
| CN112877406B (en) * | 2021-01-20 | 2022-03-18 | 南京师范大学 | Preparation method and application of organic framework material taking Ce as metal center |
| CN115032252B (en) * | 2022-04-28 | 2023-04-07 | 江南大学 | Electrochemical sensing analysis method for detecting ochratoxin A |
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